| Background: The apoptosis of hepatic stellate cells (HSCs) is an important mechanism of amelioration of liver fibrosis, while the apoptosis of liver cells plays an important role in promoting liver fibrosis. There is now considerable interest in determing the molecular events that regulate HSCs apoptosis and the discovery of drugs that will stimulate HSCs apoptosis in a selective manner. Sulfasalazine was synthesized in 1942 to combine an antibiotic, sulfapyridine, and an anti-inflammatory agent, 5-aminosalicylic acid. It was effective for treating inflammatory bowel disease (IBD). Recently, sulfasalazine has been shown to selectively induce cell apoptosis, but its mechanisms are unclear. There are only a little data concerning the effects of sulfasalazine on apoptosis in hepatic stellate cells and hepatocytes. The aim of study is to investigate the effects of sulfasalazine on proliferation and apoptosis of HSC-T6 and L02 cells and its mechanisms.Methods:1. The selection of the optimized concentration and treatment time Groups in experiment: the concentrations of sulfasalazine: 0.125, 0.25, and 0.5,1,2,4,8,16 mmol/L; treatment time: 12, 24 and 48 hour.Experiment method: The effect of sulfasalazine on proliferation of HSC-T6 was detected by CCK-8. 2. The effect of sulfasalazine on proliferation and apoptosis of HSC-T6 and L02Groups in experiment: normal control group; 0.1% DMSO control group; sulfasalazine treated group (0.5, 1, 2 mmol/L).Experiment method: the proliferation of HSC-T6 and L02 was detected by CCK-8; and the apoptosis of them was detected by ANNEXIN V-FITC/PI and AO/EB stain.3. The active drug ingredient of inducing apoptosis of HSC-T6Groups in experiment: normal control group;0.1% DMSO control group; sulfasalazine treated group(0.5,1,2mmol/L);5-aminosalicylic acid treated group(0.5,1,2mmol/L);sulfapyridine treated group(0.5, 1, 2mmol/L); 5-aminosalicylic acid(2mmol/L)+ sulfapyridine (2mmol/L)treated group.Experiment method: the proliferation of HSC-T6 was detected by CCK-8; and the apoptosis of it was detected by ANNEXIN V-FITC/PI.4. Its possible mechanisms that sulfasalazine selectively induce apoptosis of HSC-T64.1 Effect of sulfasalazine and its compounds on IKKαand IkBαphosphorylation levels in HSC-T6. Groups in experiment: normal control group; TNF-αtreated group (150U/mL); DMSO+TNF-αtreated group; sulfasalazine (0.5, 1, 2mmol/L) +TNF-αtreated group; 5-aminosalicyliacid (2mmol/L) +TNF-αtreated group; sulfapyridine (2mmol/L) + TNF-αtreated group.Experiment method: The expression levels of phospho-IKKαand phospho-I?Bαwere detected by Western blot. 4.2 Effect of sulfasalazine and its compounds on NF-kB p65 level in HSC-T6Groups in Experiment: normal control group; TNF-αtreated group(150U/mL) ;DMSO+ TNF-αtreated group; sulfasalazine(2mmol/L )+ TNF-αtreated group;5-aminosalicylic acid(2mmol/L )+ TNF-αtreated group; sulfapyridine(2mmol/L) +TNF-αtreated group.Experiment method: The expression level of NF-?B P65 was detected by Western blot; the nuclear translocation of HSC-T6 P65 was observed with Laser confocal microscope.Results:1. Compared with normal control group, the proliferation of HSC-T6 was significantly inhibited by sulfasalazine in a time and dose-dependant manner, when the concentration of sulfasalazine in the medium reached a certain level range (0.5, 1, 2, 4, 8,16mmol/L). The IC50 concentrations of inhibiting proliferation of HSC-T6 in the 12, 24, 48 hour were 6.53, 2.84, 1.5mmol/L respectively.We selected 1/3~2/3 IC50 concentrations which close to the 24 hour treated time (0.5, 1, 2mmol/L) to following experiment.2. The apoptosis rate of HSC-T6 treated with sulfasalazine (0.5, 1, 2 mmol/L) for 24 hour was 6.96±0.39%, 12.09±1.56% and 16.72±2.98% respectively, and it was 1.74±0.31%, 3.42±0.41% in normal control group and the DMSO treated group. The typical morphological changes of apoptosis were visualised by AO/EB stain in treatment of HSC-T6 with sulfasalazine.3. No significant effect was observed for either proliferation or apoptosis on L02 cells which were treated with sulfasalazine, compared with normal control group, DMSO treated group. No significant apoptosis cell was observed by AO/EB stain in treatment of L02 cells with sulfasalazine.4. After been treated by 5-aminosalicylic acid or sulfapyridine for 24 hours, there was no difference both in proliferation and apoptosis compared with normal control group, DMSO treated group.5. The expressions of phospho-IKKαand phospho-I?Bαwere respectively attenuated from TNF-α+DMSO treated group to TNF-α+ sulfasalazine(0.5,1,2mmol/L) treated group detected by Western blot.But there was no difference between TNF-α+DMSO treated group,TNF-α+ 5-aminosalicylic acid treated group, and TNF-α+ sulfapyridine treated group (P>0.05).6. The cell nuclear extracts expression of NF-?B P65 was respectively attenuated from DMSO +TNF-αtreated group, TNF-α+ sulfasalazine treated group to normal control group detected by Western blot.But there was no difference between TNF-α+DMSO treated group,TNF-α+ 5-aminosalicylic acid treated group and TNF-α+ sulfapyridine treated group (P>0.05).7. The expression of NF-?B P65 was mainly in cytoplasm in normal control group, after incubation with TNF-αfor 60 min, NF-?B P65 was markedly concentrated in the nucleus of HSC-T6.After incubation with sulfasalazine for 30 min, and NF-?B p65 was exclusively expressed in the cytoplasm of HSC-T6. Pretreated with 2mmol/L sulfasalazine 30min followed by stimulation with TNF-a 150 U/ml 60min, NF-?B P65 was markedly decreased in the nucleus of HSC-T6.Conclusions:1. Sulfasalazine can inhibit proliferation of HSC-T6 cells and stimulate HSC-T6 cells apoptosis in selective manner. However, 5-aminosalicylic acid or sulfapyridine can not affect HSC-T6 cells both in proliferation and apoptosis.2. Sulfasalazine is a selective inhibitor of NF-?B activation, our study suggests that this property is due to the ability of sulfasalazine to interfere with IKK phosphorylation and prevent nuclear translocation of NF-?B, where the Rel / NF-?B/I?B/ IKK pathway plays an important role in HSCs survival.
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